The effect of α‐phenyl‐tert‐butyl nitrone (PBN) on free radical formation in transient focal ischaemia measured by microdialysis and 3,4‐dihydroxybenzoate formation

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Abstract
α‐phenyl‐tert–butyl nitrone (PBN) reduces infarct size, improves recovery of brain energy metabolism and delays the secondary increase in extracellular potassium after focal ischaemia, presumably by trapping OH radicals. We investigated the effect of PBN on the formation of 3,4‐dihydroxybenzoic acid (3,4‐DHBA) as a measure of OH radical formation, during and following middle cerebral artery occlusion (MCAO). Rats, subjected to 2 h of ischaemia followed by 3 h of recirculation, were injected with either vehicle or PBN (100 mg kg–1 i.p.) prior to MCAO or immediately after recirculation, respectively. The in vivo microdialysis technique was used to collect samples for analysis of 3,4‐DHBA by HPLC. The basal levels of 3,4‐DHBA were 56–77 nmol L–1 in the four groups. During ischaemia, the formation of 3,4‐DHBA decreased by about 50% in all groups. Upon recirculation, a 3‐fold rise in 3,4‐DHBA formation was seen. At 2 h of recirculation the mean value of 3,4‐DHBA in the pretreated, vehicle‐injected animals was 125 ± 18 nmol L–1 and in the PBN‐injected 145 ± 48 nmol L–1, respectively. When the animals were treated after MCAO either with vehicle or PBN the values at 2 h recirculation were 155 ± 148 and 189 ± 145 nmol L–1, respectively. No statistically significant difference between vehicle‐ and PBN‐injected groups was seen. We conclude that during reperfusion following MCAO, hydroxyl radical formation increases. The increase is not ameliorated by PBN which suggests that PBN does not protect the brain by a general scavenging of OH radicals, although tissue specific actions cannot be excluded.